Streptococcus neumoniae kills more people in the U.S. than all other vaccine-preventable diseases combined and causes over 1.6 million deaths/year worldwide. The native pneumococcal vaccine currently used in adults consists of capsular polysaccharides (PPS) derived from 23 different serotypes and therefore provides broad coverage against invasive disease. Nonetheless, protection eventually wanes as antibody titers diminish, typically by 10 years post-vaccination. Unfortunately, PPS boosting does not induce recall responses. This is typical of most polysaccharide (Ps) antigens. This is a significant concern given the potential for diminished protection against pneumococcal infections. PPS-protein conjugate vaccines do not effectively boost or yield superior titers to native PPS in adults, in contrast to what is observed in young children. This, along with the high cost and limited serotype coverage of conjugate vaccines, suggests that alternative strategies are needed to enhance PPS vaccine efficacy. To improve native Ps vaccines, it is imperative to develop a mechanistic understanding of the factors preventing Ps-specific antibody recall responses. Our data supports that memory B cells are generated in response to Ps antigens but that they are largely unresponsive when antigen is re- encountered following infection or revaccination. Antigen-specific IgG may play a part by suppressing Ps- specific IgM recall responses. However, our data supports additional mechanisms of suppression contribute to not only to impaired IgM, but IgG, boosting to Ps antigens. In this proposal, we will test the hypothesis that active mechanisms of receptor-mediated immune suppression limit Ps-specific memory B cell generation and functional responsiveness to secondary antigen encounter. In addition, we will test the hypothesis that strong danger signals supplied through toll-like (TLR) and C-type lectin receptors (CLR) can overcome this suppression. In Aim 1, we will examine the B cell-intrinsic and -extrinsic roles for B7:CD28 superfamily inhibitory receptors and ligands in suppressing Ps-specific memory B cell formation and effector function. In Aim 2, we will investigate mechanisms by which Ps-specific antibody suppresses boosting. In Aim 3, we will examine the extent to which TLR4/RP105 and Mincle agonists synergize to promote Ps-specific memory B cell generation and functional responsiveness during boosting. We will examine the mechanisms by which this is achieved, including the extent to which these agonists overcome the inhibitory pathways examined in Aims 1 and 2. Completion of these studies is expected to provide critical knowledge regarding the mechanisms responsible for suppressed secondary antibody responses to native Ps as well as the feasibility of using TLR and CLR-based adjuvant combinations to overcome this barrier to effective vaccination. Our results are expected to have a significant impact on the use of prime-boost strategies employing native Ps, as well as the future design of more affordable and efficacious Ps-based vaccines with the highest possible serotype coverage.